Early work demonstrated that 2-butoxyethanol (BE) causes acute hemolytic anemia in rats. Treatment of rats with BE daily (125 mg/kg/day) for three consecutive days resulted in a time dependent increase in the hemolysis of erythrocytes. However, when daily treatment continued beyond 3 days, the number of erythrocytes started to increase and approached the pretreatment level within 10 days despite continued daily dosing, suggesting tolerance development to the hemolytic effect of BE. To investigate the mechanism of tolerance development, rats were treated with 125 mg BE/kg/day for 3 days and allowed to recover (with no treatment) for 7 days. Control rats were treated with water similarly. At the end of this recovery period, rats were treated with 125 or 250 mg BE/kg and the hematology profiles were assessed. A significant decline in the sensitivity of pretreated rats compared to vehicle controls were observed. Furthermore, incubation of blood obtained from the recovered rats with the hematotoxic metabolite of BE, 2-butoxyacetic acid (BAA), in vitro indicated that there was a decline in the sensitivity of these erythrocytes as well. In another series of studies, the effect of BE on erythrocytes morphology in vivo and the effect of BAA on human and rat erythrocytes in vitro was investigated. Both BE and BAA caused stomatocytosis and vesiculation in rat erythrocytes. In contrast, no such effects were seen on human erythrocytes.